Background
Vector control is a major component of the global strategy for malaria control which aims to prevent parasite transmission mainly through interventions targeting adult anopheline vectors [
1]. Ongoing strategies rely heavily on the use of safe and effective insecticides through indoor residual spraying (IRS) or insecticide-treated nets (ITNs). Successful implementation of these strategies requires sound knowledge of vectors distributions, biology and susceptibility to available insecticide compounds. Currently, the National Malaria Control Programme (NMCP) of Chad is promoting large scale use of ITNs as the main vector control tool but little is known on the vectors responsible for malaria transmission in the country. Back to the 1960's, thirteen anopheline species were recorded [
2], with mosquitoes from the
Anopheles gambiae complex, including
An. gambiae s.s. and
Anopheles arabiensis being the most abundant and widespread. However, these data have never been updated and susceptibility to insecticides used for net treatment has never been assessed in Chad.
In many African countries,
An. gambiae s.l. is developing resistance to all classes of insecticides used for mosquito control. Among these, pyrethroids are the only option for nets treatment due to their relative safety for humans at low dosage, excito-repellent properties, rapid rate of knock-down and killing effects [
3]. The emergence and rapid spread of pyrethroid resistance in
An. gambiae complex populations may be a threat for the sustainability of malaria vector control activities across Africa. Comprehensive knowledge of the factors underlying resistance is needed for the implementation of efficient vector control programmes including resistance management strategies. This raises the need for countrywide and regular surveys for monitoring the insecticide susceptibility status of major vectors, detecting resistance genes and assessing their implications on vector control activities [
4]. Anopheline mosquitoes exhibit two major mechanisms of pyrethroids resistance: increased insecticide detoxification (metabolic resistance) and target site insensitivity [
5]. Metabolic resistance to pyrethroids is usually associated with increased oxidases and esterases activity [
6] while target site insensitivity results from point mutations in the voltage-gated sodium channel gene [
7,
8]. The latter mechanism also termed
kdr (knock-down resistance) induces cross resistance to DDT. Two alternative
kdr mutations have been described in
An. gambiae s.l. populations, resulting in either a leucine-phenylalanine (L1014F), or a leucine-serine (L1014S) mutation at amino acid position 1014 of the sodium channel [
7,
8]. The former is widely distributed in the M and S forms of
An. gambiae in West Africa, and the latter, originally described from Kenya, has been recently found in the S form of
An. gambiae from Central Africa (see [
9] for a recent review).
Metabolic and target site resistance have been found alone or occurring together in many countries bordering Chad as Cameroon [
10‐
12], Central African Republic [
13], Nigeria [
14‐
16] and Sudan [
17,
18]. Pyrethroid insecticides are commonly used in Chad for crop protection and, more recently, for malaria vector control through large-scale ITN distribution programmes (NMCP, unpublished reports). In such a context, the emergence and spread of pyrethroid resistance is extremely likely in Chad. The present survey was hence designed to investigate the susceptibility of
An. gambiae s.l. to the four classes of insecticides used in public health. It was carried out in Western Chad, in three health districts where ITNs distribution programmes are being implemented.
Discussion
This study revealed the existence of permethrin and deltamethrin resistance in several
An. gambiae s.l. populations from south-western Chad. Full susceptibility to malathion and bendiocarb was recorded in all samples tested, except in Mbéré, a village of the health district of Guelendeng where some level of tolerance to bendiocarb was observed. Full susceptibility to all insecticides tested was only observed with the sample from the urban area of Guelendeng. These various levels of insecticide susceptibility may reflect differential insecticide selection pressure exerted on field mosquito populations. The health district of Kélo is situated in a cotton growing area with intensive use of pyrethroid and organophosphorous compounds. The situation is quite different in Guelendeng and Bongor since chemical crop protection is not common in these areas. In fact, cotton cultivation in these two districts have ceased since the civil unrest in 1979. Before this date, organochlorines including DDT and dieldrin were regularly sprayed in the cotton fields. These regions experienced also chemical treatment against locust in the seventies [
26]. Dieldrin, DDT, HCH and pyrethroids have also been used in the irrigated rice fields in Bongor but due to a lack of archives, appropriate information regarding doses and frequencies of treatment is not available. Some of these insecticides may have persisted in the environment, leading to subsequent selection of various resistance mechanisms in vector populations. In many African countries, resistance to pyrethroids has been attributed to extensive use of these compounds in agriculture [
27,
28], resistance levels being more important in cotton cultivation areas than in others agricultural schemes [
12,
29,
30]. This is consistent with pyrethroid resistance being detected in the cotton growing area of Kélo. However, increased tolerance to pyrethroids in Bongor and, to a lesser extent in Guelendeng suggests additional selection pressure. Insecticide use for vector control interventions and/or personal protection against nuisances has been suggested as an additional putative source of selective pressure for pyrethroid resistance in malaria vectors, especially in urban cities and irrigated areas [
30‐
32]. Indeed, in Bongor and adjacent areas, in addition to the recently introduced ITNs, coils and bomb sprays are frequently used for personal protection [
33]. Further studies involving social scientists, chemical ecologists and environmental biologists would be needed to document the amount, frequency and diversity of insecticides used in these areas in order to explore in greater details the putative selective pressures leading to the selection of insecticide resistance in malaria vectors.
Anopheles arabiensis was found to be the predominant species of the
An. gambiae complex in the study area. It constituted nearly 100% of the samples in Guelendeng and Bongor where the mean annual rainfall is the lowest.
Anopheles gambiae s.s. was more abundant southwards in Kélo, where its two molecular forms were found to occur together with
An. arabiensis, the latter still being predominant whatever the village. The species distribution within the
An. gambiae complex is consistent with literature data for whole sub-Saharan Africa [
34,
35].
One of the main findings of the present study is the first report of the L1014F
kdr mutation in wild
An. gambiae populations from Chad. The resistant allele was found only in the S molecular form of
An. gambiae s.s. in all villages sampled in the health district of Kélo. This finding is of paramount importance given recent evidences that
kdr-based resistance mechanisms can jeopardize the efficacy of ITNs and IRS [
36,
37]. However, the strength of the correlation between the genotype at the
kdr locus and the expression of insecticide resistance has been shown to vary in different genetic backgrounds an under different ecological settings [
38,
39]. Integrated investigations, using a more complete range of methodologies which allow detection of target sites mutations along with exploration of metabolic detoxification should be implemented in order to provide a more comprehensive overview of the genetic bases and mechanisms responsible for the resistance phenotype detected in these mosquito populations. Currently, the development of more sensitive tools is underway, greatly facilitated by recent advances in genomics. Some of these tools have been successfully tested, leading to more comprehensive knowledge of the molecular mechanisms of metabolic resistance [
40,
41]. Nevertheless, this new report of the L1014F mutation in
An. gambiae from Chad further witnesses the ongoing spread of
kdr mutations in Africa [
9].
Absence of the
kdr mutations in all surviving
An. arabiensis suggests alternative resistance mechanisms, probably of metabolic origin are at play. This finding is not surprising because although both
kdr mutations have been documented to occur in
An. arabiensis, they are usually very scarce and are found floating at much lower frequencies than in its sibling,
An. gambiae [
12,
17,
32,
42,
43]. Increased monooxygenase activities were reported to be associated with pyrethroid resistance in major malaria vectors including
An. arabiensis in East and Central Africa [
6,
11,
44]. More recently, some general mechanisms occurring through a set of constitutively over-expressed genes with ability to control oxidative stress and other broad metabolic disorders was suggested to act as a general defence mechanism against deltamethrin in
An. arabiensis populations from a neighbouring area of cotton cultivation in North Cameroon [
12,
41]. Similar mechanisms might occur in the
An. arabiensis populations sampled in this study and further research into the mechanism(s) responsible for the high levels of resistance to pyrethroids occurring in western Chad are ongoing.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
CKH conceived and designed the study, coordinated its implementation in the fields, carried out laboratory procedures, analysed and interpreted data, and wrote the manuscript; MP carried out field experiments, analysed and interpreted data; PN helped with molecular processing, analysis and interpretation of data; IDG participated in the design of the study and supervised fields experiments; JE helped with data analysis and contributed in the drafting of the manuscript; ASE participated in the study design, participated in data analysis and interpretation and provided a critical review of the manuscript; FS participated in the study design, supervised fields and laboratory procedures, data analysis and interpretation, revised the manuscript and gave final approval for the version to be published. All authors read and approved the final manuscript.